Basic-liver, pancreas, and biliary tractBid activates multiple mitochondrial apoptotic mechanisms in primary hepatocytes after death receptor engagement☆
Section snippets
Mice
Mice deficient in bid were established by gene targeting and had been back-crossed to the C57BL/6 background for 12 generations.12, 13 Wild-type and bid-deficient mice used in this study were littermates and weighed about 25–30 g. They were maintained as homozygotes in a specific pathogen-free facility on the campus in compliance with the National Institutes of Health and University of Pittsburgh policies.
Establishment of primary hepatocyte culture
Murine hepatocytes were isolated using the collagenase digestion method as previously
Hepatocytes deficient in bid are refractory to anti-Fas or TNF-α
To study the mechanism by which Bid activates the mitochondria in the cell, we had set up a primary hepatocyte culture and verified the role of Bid in anti-Fas- or TNF-α—induced apoptosis in this system. Cell viability was monitored at different times after the treatment by MTT assay (Figure 1A), nuclear staining (Figure 1B), trypan blue staining, and visual inspection by phase microscopy (data not shown). Data from all these assays were comparable and showed that a majority of wild-type
Discussion
A number of studies on death receptor—induced hepatocyte apoptosis have been conducted, but some of the key questions remained unanswered, particularly those about mitochondria activation. Because the analysis can be performed in a proper cellular environment, the cell culture system provides a unique way to examine these issues, which may not be readily addressable by the animal studies or the in vitro reconstitution analysis. We thus aimed to determine the mechanism of mitochondria activation
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Supported in part by the Howard Temin Award (K01 CA 74885, National Institutes of Health), R01 CA 83817, National Institutes of Health (to X.-M.Y.), and P01 DK59340, National Institutes of Health (to J.J.L.).
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Y.Z. and W.-X.D. contributed equally to this work.